Macaque antibodies targeting Marburg virus glycoprotein induced by multivalent immunization.

Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.

Hepatitis C Virus E1E2 Structure, Diversity, and Implications for Vaccine Development.

Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein.

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

Site-directed neutralizing antibodies targeting structural sites on SARS-CoV-2 spike protein.

'Epivolve' (epitope evolution) is an innovative paratope-evolving technology using a haptenated peptide or protein immunogen as a means of directing the in vivo immune response to specifically targeted sites at a one amino acid residue resolution. Guided by protein structural analysis, Epivolve technology was tested to develop site-directed neutralizing antibodies (nAbs) in a systematic fashion against the SARS-CoV-2 Receptor Binding Domain (RBD). Thirteen solvent-exposed sites covering the ACE2 receptor-binding interface were targeted. Immunogens composed of each targeted site were used to immunize rabbits in separate cohorts. In vivo site-directed immune responses against all 13 targets were demonstrated by B cell secreted IgG and recombinant IgG testing. One site, SL13 (Y505) which mutates from tyrosine to histidine in the SARS-CoV-2 Omicron variant, was chosen as a proof-of-concept (PoC) model for further functional monoclonal antibody development. Epivolve technology demonstrated the capabilities of generating pan-variant antibodies and nAbs against the SARS-CoV-2 primary strain and the Omicron variant.

Towards Improved Identification of Vertebral Fractures in Routine Computed Tomography (CT) Scans: Development and External Validation of a Machine Learning Algorithm.

Vertebral fractures (VFs) are the hallmark of osteoporosis, being one of the most frequent types of fragility fracture and an early sign of the disease. They are associated with significant morbidity and mortality. VFs are incidentally found in one out of five imaging studies, however, more than half of the VFs are not identified nor reported in patient computed tomography (CT) scans. Our study aimed to develop a machine learning algorithm to identify VFs in abdominal/chest CT scans and evaluate its performance. We acquired two independent data sets of routine abdominal/chest CT scans of patients aged 50 years or older: a training set of 1011 scans from a non-interventional, prospective proof-of-concept study at the Universitair Ziekenhuis (UZ) Brussel and a validation set of 2000 subjects from an observational cohort study at the Hospital of Holbaek. Both data sets were externally reevaluated to identify reference standard VF readings using the Genant semiquantitative (SQ) grading. Four independent models have been trained in a cross-validation experiment using the training set and an ensemble of four models has been applied to the external validation set. The validation set contained 15.3% scans with one or more VF (SQ2-3), whereas 663 of 24,930 evaluable vertebrae (2.7%) were fractured (SQ2-3) as per reference standard readings. Comparison of the ensemble model with the reference standard readings in identifying subjects with one or more moderate or severe VF resulted in an area under the receiver operating characteristic curve (AUROC) of 0.88 (95% confidence interval [CI], 0.85-0.90), accuracy of 0.92 (95% CI, 0.91-0.93), kappa of 0.72 (95% CI, 0.67-0.76), sensitivity of 0.81 (95% CI, 0.76-0.85), and specificity of 0.95 (95% CI, 0.93-0.96). We demonstrated that a machine learning algorithm trained for VF detection achieved strong performance on an external validation set. It has the potential to support healthcare professionals with the early identification of VFs and prevention of future fragility fractures. © 2023 UCB S.A. and The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Prospects for developing an Hepatitis C virus E1E2-based nanoparticle vaccine.

Globally, more than 58 million people are chronically infected with Hepatitis C virus (HCV) with 1.5 million new infections occurring each year. An effective vaccine for HCV is therefore a major unmet medical and public health need. Since HCV rapidly accumulates mutations, vaccines must elicit the production of broadly neutralising antibodies (bnAbs) in a reproducible fashion. Decades of research have generated a number of HCV vaccine candidates. Based on the available data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice, but robust induction of humoral and cellular responses leading to virus neutralisation has not yet been achieved. One issue that has arisen in developing an HCV vaccine (and many other vaccines as well) is the platform used for antigen delivery. The majority of viral vaccine trials have employed subunit vaccines. However, subunit vaccines often have limited immunogenicity, as seen for HCV, and thus multiple formats must be examined in order to elicit a robust anti-HCV immune response. Nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both arms of the immune system. This review discusses the potential for development of a nanoparticle-based HCV E1E2 vaccine, with an emphasis on the potential benefits of such an approach along with the major challenges facing the incorporation of E1E2 into nanoparticulate delivery systems and how those challenges can be addressed.

Structure of engineered hepatitis C virus E1E2 ectodomain in complex with neutralizing antibodies.

Hepatitis C virus (HCV) is a major global health burden as the leading causative agent of chronic liver disease and hepatocellular carcinoma. While the main antigenic target for HCV-neutralizing antibodies is the membrane-associated E1E2 surface glycoprotein, the development of effective vaccines has been hindered by complications in the biochemical preparation of soluble E1E2 ectodomains. Here, we present a cryo-EM structure of an engineered, secreted E1E2 ectodomain of genotype 1b in complex with neutralizing antibodies AR4A, HEPC74, and IGH520. Structural characterization of the E1 subunit and C-terminal regions of E2 reveal an overall architecture of E1E2 that concurs with that observed for non-engineered full-length E1E2. Analysis of the AR4A epitope within a region of E2 that bridges between the E2 core and E1 defines the structural basis for its broad neutralization. Our study presents the structure of an E1E2 complex liberated from membrane via a designed scaffold, one that maintains all essential structural features of native E1E2. The study advances the understanding of the E1E2 heterodimer structure, crucial for the rational design of secreted E1E2 antigens in vaccine development.

Glycosylation shapes the efficacy and safety of diverse protein, gene and cell therapies.

Over recent decades, therapeutic proteins have had widespread success in treating a myriad of diseases. Glycosylation, a near universal feature of this class of drugs, is a critical quality attribute that significantly influences the physical properties, safety profile and biological activity of therapeutic proteins. Optimizing protein glycosylation, therefore, offers an important avenue to developing more efficacious therapies. In this review, we discuss specific examples of how variations in glycan structure and glycoengineering impacts the stability, safety, and clinical efficacy of protein-based drugs that are already in the market as well as those that are still in preclinical development. We also highlight the impact of glycosylation on next generation biologics such as T cell-based cancer therapy and gene therapy.

Fluorine-Functionalized Polyphosphazene Immunoadjuvant: Synthesis, Solution Behavior and In Vivo Potency.

The inclusion of fluorine motifs in drugs and drug delivery systems is an established tool for modulating their biological potency. Fluorination can improve drug specificity or boost the vehicle's ability to cross cellular membranes. However, the approach has yet to be applied to vaccine adjuvants. Herein, the synthesis of fluorinated bioisostere of a clinical stage immunoadjuvant-poly[di(carboxylatophenoxy)phosphazene], PCPP-is reported. The structure of water-soluble fluoropolymer-PCPP-F, which contains two fluorine atoms per repeat unit-was confirmed using 1H, 31P and 19F NMR, and its molecular mass and molecular dimensions were determined using size-exclusion chromatography and dynamic light scattering. Insertion of fluorine atoms in the polymer side group resulted in an improved solubility in acidic solutions and faster hydrolytic degradation rate, while the ability to self-assemble with an antigenic protein, lysozyme-an important feature of polyphosphazene vaccine adjuvants-was preserved. In vivo assessment of PCPP-F demonstrated its greater ability to induce antibody responses to Hepatitis C virus antigen when compared to its non-fluorinated counterpart. Taken together, the superior immunoadjuvant activity of PCPP-F, along with its improved formulation characteristics, demonstrate advantages of the fluorination approach for the development of this family of macromolecular vaccine adjuvants.

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Implementation of the OMERACT Psoriatic Arthritis Magnetic Resonance Imaging Scoring System in a randomized phase IIb study of abatacept in psoriatic arthritis.

OBJECTIVES: To investigate if the OMERACT PsA MRI Scoring System (PsAMRIS), including a novel total inflammation score, shows sensitivity to change with an agent (abatacept) known to impact clinical outcomes in PsA. METHODS: We performed a post hoc analysis of a randomized phase IIb study of abatacept in patients with PsA and inadequate DMARD response. Participants received one of three abatacept dosing regimens [ABA3, ABA10 or ABA30/10 mg/kg (30 mg/kg switched to 10 mg/kg after two doses)] or placebo until day 169, then ABA10 through day 365. MRIs at baseline and days 85, 169 and 365 were centrally evaluated by two readers blinded to chronological order and treatment arm. Synovitis, osteitis, tenosynovitis, periarticular inflammation, bone erosions, joint space narrowing and bone proliferation were assessed using the PsAMRIS. A novel total inflammation score was tested. RESULTS: MRIs for 123 patients were included. On day 169, ABA10 and ABA30/10 significantly reduced MRI synovitis and tenosynovitis, respectively, vs placebo [differences -0.966 (P = 0.039) and -1.652 (P = 0.014), respectively]. Synovitis in the placebo group increased non-significantly from baseline to day 169, total inflammation and tenosynovitis decreased non-significantly and all measures improved significantly after a switch to ABA10 [-1.019, -0.940, -2.275 (P < 0.05), respectively, day 365 vs day 169]. Structural outcomes changed minimally across groups. CONCLUSION: Adults with PsA receiving ABA10 and ABA30/10 demonstrated significant resolution of inflammatory components of disease, confirmed by MRI, with synovitis and tenosynovitis improvements consistent with previously reported clinical responses for these doses. Results indicate that a reduction in OMERACT PsAMRIS inflammation scores may provide proof of tissue-level efficacy in PsA clinical trials. REGISTRATION: ClinicalTrials.gov (https://clinicaltrials.gov), NCT00534313.

An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development.

BACKGROUND & AIMS: Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS: We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS: Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS: Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.

Skin Vaccination with Ebola Virus Glycoprotein Using a Polyphosphazene-Based Microneedle Patch Protects Mice against Lethal Challenge.

Ebolavirus (EBOV) infection in humans is a severe and often fatal disease, which demands effective interventional strategies for its prevention and treatment. The available vaccines, which are authorized under exceptional circumstances, use viral vector platforms and have serious disadvantages, such as difficulties in adapting to new virus variants, reliance on cold chain supply networks, and administration by hypodermic injection. Microneedle (MN) patches, which are made of an array of micron-scale, solid needles that painlessly penetrate into the upper layers of the skin and dissolve to deliver vaccines intradermally, simplify vaccination and can thereby increase vaccine access, especially in resource-constrained or emergency settings. The present study describes a novel MN technology, which combines EBOV glycoprotein (GP) antigen with a polyphosphazene-based immunoadjuvant and vaccine delivery system (poly[di(carboxylatophenoxy)phosphazene], PCPP). The protein-stabilizing effect of PCPP in the microfabrication process enabled preparation of a dissolvable EBOV GP MN patch vaccine with superior antigenicity compared to a non-polyphosphazene polymer-based analog. Intradermal immunization of mice with polyphosphazene-based MN patches induced strong, long-lasting antibody responses against EBOV GP, which was comparable to intramuscular injection. Moreover, mice vaccinated with the MN patches were completely protected against a lethal challenge using mouse-adapted EBOV and had no histologic lesions associated with ebolavirus disease.

Structural and Biophysical Characterization of the HCV E1E2 Heterodimer for Vaccine Development.

An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.

Immunopotentiating and Delivery Systems for HCV Vaccines.

Development of preventive vaccines against hepatitis C virus (HCV) remains one of the main strategies in achieving global elimination of the disease. The effort is focused on the quest for vaccines capable of inducing protective cross-neutralizing humoral and cellular immune responses, which in turn dictate the need for rationally designed cross-genotype vaccine antigens and potent immunoadjuvants systems. This review provides an assessment of the current state of knowledge on immunopotentiating compounds and vaccine delivery systems capable of enhancing HCV antigen-specific immune responses, while focusing on the synergy and interplay of two modalities. Structural, physico-chemical, and biophysical features of these systems are discussed in conjunction with the analysis of their in vivo performance. Extreme genetic diversity of HCV-a well-known hurdle in the development of an HCV vaccine, may also present a challenge in a search for an effective immunoadjuvant, as the effort necessitates systematic and comparative screening of rationally designed antigenic constructs. The progress may be accelerated if the preference is given to well-defined molecular immunoadjuvants with greater formulation flexibility and adaptability, including those capable of spontaneous self-assembly behavior, while maintaining their robust immunopotentiating and delivery capabilities.

Engineering subtilisin proteases that specifically degrade active RAS.

We describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

Intracellular Delivery of Active Proteins by Polyphosphazene Polymers.

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.